中华检验医学杂志
中華檢驗醫學雜誌
중화검험의학잡지
CHINESE JOURNAL OF LABORATORY MEDICINE
2011年
4期
327-332
,共6页
杨卓%龙美娟%王斐%陈倩%赵保健%郭野%黄媛%苏秀兰%张旭%崔巍
楊卓%龍美娟%王斐%陳倩%趙保健%郭野%黃媛%囌秀蘭%張旭%崔巍
양탁%룡미연%왕비%진천%조보건%곽야%황원%소수란%장욱%최외
癌,非小细胞肺%结直肠肿瘤%受体,表皮生长因子%原癌基因蛋白质类%突变%色谱法,高压液相
癌,非小細胞肺%結直腸腫瘤%受體,錶皮生長因子%原癌基因蛋白質類%突變%色譜法,高壓液相
암,비소세포폐%결직장종류%수체,표피생장인자%원암기인단백질류%돌변%색보법,고압액상
Carcinoma,non-small-cell lung%Colorectal neoplasms%Receptor,epidermal growth factor%Proto-oncogene proteins%Mutation%Chromatography,high pressure liquicl
目的 建立一种REDE-DHPLC检测外周血肿瘤游离核酸EGFR、KRAS基因突变的方法,并探讨其临床应用价值.方法 采用限制性内切酶Mse Ⅰ、Msc Ⅰ、BstN Ⅰ和BglⅠ分别切断EGFR基因第19、21号外显子和KRAS基因第12、13号密码子的野生型片段以富集突变片段,建立检测血浆EGFR和KRAS基因突变的REDE-DHPLC法,并采用50%、10%、5%、1%和0.1%稀释度的质粒标准品评价REDE-DHPLC法和传统DHPLC法的检测灵敏度.然后,采用REDE-DHPLC法检测120例NSCLC和120例结直肠癌患者血浆和石蜡组织标本中的EGFR和KRAS基因突变.同时,用传统DHPLC法和测序法进行平行检测,以评价REDE-DHPLC法检测NSCLC和结直肠癌患者血浆中2个基因突变的诊断效能.结果 REDE-DHPLC法对EGFR和KRAS基因4个突变位点检测的灵敏度均达0.1%,传统DHPLC法检测灵敏度均为1.0%,REDE-DHPLC法对含0.1%突变的质粒标准品重复2～3次检测均为阳性.REDE-DHPLC法、传统DHPLC法和测序法检测120例NSCLC患者血浆EGFR基因总突变率分别为27.5%、16.7%和12.5%,检测120例结直肠癌患者血浆KRAS基因总突变率分别为38.3%、25.8%和16.7%.REDE-DHPLC法检测EGFR和KRAS基因总突变率均高于传统DHPLC法(x2值分别为4.092、4.301,P均＜0.05)和测序法(x2值分别为8.438、14.127,P均＜0.05);将REDE-DHPLC法检测EGFR和KRAS基因突变与传统DHPLC法相比,敏感度均为100%(20/20,31/31),特异度分别为87.0%(87/100)和83.2%(74/89);与测序法相比,敏感度均为100%(15/15,20/20),特异度分别为82.9%(87/105)和74.0%(74/100).REDE-DHPLC法与传统DHPLC法检测EGFR、KRAS基因突变的符合率分别为89.2%(107/120,Kappa=0.690,P＜0.05)和87.5%(105/120,Kappa=0.718,P＜0.05).REDE-DHPLC检测血浆与组织标本中EGFR和KRAS基因总突变符合率分别为91.7%(33/36,Kappa=0.939,P＜0.05)和90.2%(46/51,Kappa=0.914,P＜0.05).结论 REDE-DHPLC法灵敏度和特异性高,结果易判读,且可有效避免纯合点突变漏检,有望成为临床实验室外周血EGFR和KRAS基因突变检测的推广方法.
目的 建立一種REDE-DHPLC檢測外週血腫瘤遊離覈痠EGFR、KRAS基因突變的方法,併探討其臨床應用價值.方法 採用限製性內切酶Mse Ⅰ、Msc Ⅰ、BstN Ⅰ和BglⅠ分彆切斷EGFR基因第19、21號外顯子和KRAS基因第12、13號密碼子的野生型片段以富集突變片段,建立檢測血漿EGFR和KRAS基因突變的REDE-DHPLC法,併採用50%、10%、5%、1%和0.1%稀釋度的質粒標準品評價REDE-DHPLC法和傳統DHPLC法的檢測靈敏度.然後,採用REDE-DHPLC法檢測120例NSCLC和120例結直腸癌患者血漿和石蠟組織標本中的EGFR和KRAS基因突變.同時,用傳統DHPLC法和測序法進行平行檢測,以評價REDE-DHPLC法檢測NSCLC和結直腸癌患者血漿中2箇基因突變的診斷效能.結果 REDE-DHPLC法對EGFR和KRAS基因4箇突變位點檢測的靈敏度均達0.1%,傳統DHPLC法檢測靈敏度均為1.0%,REDE-DHPLC法對含0.1%突變的質粒標準品重複2～3次檢測均為暘性.REDE-DHPLC法、傳統DHPLC法和測序法檢測120例NSCLC患者血漿EGFR基因總突變率分彆為27.5%、16.7%和12.5%,檢測120例結直腸癌患者血漿KRAS基因總突變率分彆為38.3%、25.8%和16.7%.REDE-DHPLC法檢測EGFR和KRAS基因總突變率均高于傳統DHPLC法(x2值分彆為4.092、4.301,P均＜0.05)和測序法(x2值分彆為8.438、14.127,P均＜0.05);將REDE-DHPLC法檢測EGFR和KRAS基因突變與傳統DHPLC法相比,敏感度均為100%(20/20,31/31),特異度分彆為87.0%(87/100)和83.2%(74/89);與測序法相比,敏感度均為100%(15/15,20/20),特異度分彆為82.9%(87/105)和74.0%(74/100).REDE-DHPLC法與傳統DHPLC法檢測EGFR、KRAS基因突變的符閤率分彆為89.2%(107/120,Kappa=0.690,P＜0.05)和87.5%(105/120,Kappa=0.718,P＜0.05).REDE-DHPLC檢測血漿與組織標本中EGFR和KRAS基因總突變符閤率分彆為91.7%(33/36,Kappa=0.939,P＜0.05)和90.2%(46/51,Kappa=0.914,P＜0.05).結論 REDE-DHPLC法靈敏度和特異性高,結果易判讀,且可有效避免純閤點突變漏檢,有望成為臨床實驗室外週血EGFR和KRAS基因突變檢測的推廣方法.
목적 건립일충REDE-DHPLC검측외주혈종류유리핵산EGFR、KRAS기인돌변적방법,병탐토기림상응용개치.방법 채용한제성내절매Mse Ⅰ、Msc Ⅰ、BstN Ⅰ화BglⅠ분별절단EGFR기인제19、21호외현자화KRAS기인제12、13호밀마자적야생형편단이부집돌변편단,건립검측혈장EGFR화KRAS기인돌변적REDE-DHPLC법,병채용50%、10%、5%、1%화0.1%희석도적질립표준품평개REDE-DHPLC법화전통DHPLC법적검측령민도.연후,채용REDE-DHPLC법검측120례NSCLC화120례결직장암환자혈장화석사조직표본중적EGFR화KRAS기인돌변.동시,용전통DHPLC법화측서법진행평행검측,이평개REDE-DHPLC법검측NSCLC화결직장암환자혈장중2개기인돌변적진단효능.결과 REDE-DHPLC법대EGFR화KRAS기인4개돌변위점검측적령민도균체0.1%,전통DHPLC법검측령민도균위1.0%,REDE-DHPLC법대함0.1%돌변적질립표준품중복2～3차검측균위양성.REDE-DHPLC법、전통DHPLC법화측서법검측120례NSCLC환자혈장EGFR기인총돌변솔분별위27.5%、16.7%화12.5%,검측120례결직장암환자혈장KRAS기인총돌변솔분별위38.3%、25.8%화16.7%.REDE-DHPLC법검측EGFR화KRAS기인총돌변솔균고우전통DHPLC법(x2치분별위4.092、4.301,P균＜0.05)화측서법(x2치분별위8.438、14.127,P균＜0.05);장REDE-DHPLC법검측EGFR화KRAS기인돌변여전통DHPLC법상비,민감도균위100%(20/20,31/31),특이도분별위87.0%(87/100)화83.2%(74/89);여측서법상비,민감도균위100%(15/15,20/20),특이도분별위82.9%(87/105)화74.0%(74/100).REDE-DHPLC법여전통DHPLC법검측EGFR、KRAS기인돌변적부합솔분별위89.2%(107/120,Kappa=0.690,P＜0.05)화87.5%(105/120,Kappa=0.718,P＜0.05).REDE-DHPLC검측혈장여조직표본중EGFR화KRAS기인총돌변부합솔분별위91.7%(33/36,Kappa=0.939,P＜0.05)화90.2%(46/51,Kappa=0.914,P＜0.05).결론 REDE-DHPLC법령민도화특이성고,결과역판독,차가유효피면순합점돌변루검,유망성위림상실험실외주혈EGFR화KRAS기인돌변검측적추엄방법.
Objective To establish a REDE-DHPLC method for detecting the EGFR and KRAS mutations in plasma DNA from tumor patients, and investigate its clinical significance. Methods Restriction endonucleases Mse Ⅰ , Msc Ⅰ , BstN Ⅰ and Bgl Ⅰ were used to digest the wild type fragments of exon 19,exon 21 of EGFR gene and coden 12, 13 of KRAS gene for enriching the mutation fragments, and REDE-DHPLC method was established to detect EGFR and KRAS mutations. The sensitivities of REDE-DHPLC and conventional DHPLC were analyzed by using a series of plasmids containing 50%, 10%, 5%, 1% and 0. 1% mutation genes. Then, Plasma samples and paraffin-embedded tissue samples of 120 NSCLC patients and 120 colorectal cancer patients were detected by REDE-DHPLC. Compared with conventional DHPLC and sequencing, the diagnostic efficiency of REDE-DHPLC method was evaluated by detecting the mutation status of 2 genes in plasma of NSCLC and colorectal cancer patients. Results The sensitivity values of REDE-DHPLC and conventional DHPLC for detecting mutations in 4 loci were 0. 1% and 1%respectively. Plasmid DNA containing 0.1% mutation gene was detected to be positive continually for 2 to 3 times by REDE-DHPLC. EGFR mutation rates of 120 plasma from NSCLC patients detected by REDE-DHPLC, conventional DHPLC and sequencing methods were 27. 5%, 16. 7% and 12.5% respectively, and KRAS mutation rates of 120 plasma from colorectal cancer patients were 38. 3%, 25. 8% and 16. 7%,respectively. The positive rates of EGFR and KRAS mutation detected by REDE-DHPLC were significantly higher than conventional DHPLC(x2 = 4. 092, 4. 301, all P ＜ 0. 05 ) and sequencing method (x2= 8. 438,14. 127,all P ＜ 0. 05 ). In comparison with conventional DHPLC, the sensitivities of REDE-DHPLC for detecting EGFR and KRAS mutation were 100% (20/20,31/31), the specificities were 87. 0% (87/100)and 83. 2% (74/89). In comparison with sequencing method, the sensitivities of REDE-DHPLC were 100%( 15/15,20/20), the specificities were 82.9% (87/105)and 74. 0% (74/100). The coincidence rate of the two methods for detecting EGFR and KRAS mutation were 89. 2% ( 107/120, Kappa = 0. 690, P ＜ 0. 05 ) and 87.5% ( 105/120, Kappa= 0. 718, P ＜ 0. 05 ). The Consistency of EGFR and KRAS mutation status in plasma and tissues detected by REDE-DHPLC were 91.7% (33/36, Kappa =0. 939,P ＜0. 05)and 90. 2 %(46/51, Kappa = 0. 914, P ＜ 0. 05 ), respectively. Conclusions The REDE-DHPLC method is highly sensitive and specific for detecting EGFR and KRAS mutations in plasma DNA from tumor patients. The results are easy to be interpreted without missing homozygous point mutation, which indicate that the detection of EGFR and KRAS mutations in plasma DNA by REDE-DHPLC could therefore extend to be usedin clinical laboratory.
